We analyzed the genetic linkage of nevirapine (NVP) resistance mutations and the genetic complexity of HIV-1 variants in Ugandan infants who were HIV infected despite single dose (SD) prophylaxis. Plasma samples were obtained from six HIV-infected infants who had two or more NVP resistance mutations detected by population sequencing (ViroSeq). ViroSeq PCR products were cloned and transformed, and a single-step amplification-sequencing reaction (AmpliSeq) was used to analyze NVP resistance mutations in cloned HIV-1 variants directly from bacterial colonies. Fifty clones were analyzed for each infant sample. This analysis revealed numerous NVP resistance mutations not detected by population sequencing, genetically linked NVP resistance mutations, and a high degree of genetic complexity at codons that influence NVP susceptibility.